Chemical Senses Volume 6 Number 4 1981

Chemical blockade of olfactory perception by N-methyl-formimino-methylester in albino mice. II. Light microscopicalinvestigations

B.Rehn1.2, W.Breipohl2*, C.Schmidt3, U.Schmidt3, F.Effenberger*iInstitut fiir Zoologie, University Tubingen, Tubingen; 2Institut fiir Anatomie,Universita'tsklinikum Essen, Essen; 3Institut fiir Zoologie der Universita't Bonn,Bonn; andKlnstitutfiir Organische Chemie, Universitdt Stuttgart, Stuttgart, FRG

Abstract. Evidence is given that N-methyl-formimino-methylester (MFM), a highly volatile substance,causes olfactory receptor cell degeneration in mice. The time course of this degeneration and mor-phological changes in the compartment of receptor cell progenitor cells are described. Due to the mor-phological appearance of the progenitor cells and their dynamic transformation after exposure toMFM, two different types of progenitor cells can be distinguished: (a) light-staining globose basal orblastema cells, which are thought to be real progenitor cells and to remain in the mitotic cyde forgenerating new sensory cells; and (b) dark-staining basal cells with condensed chromatin, which arequiescent. The results agree with electrophysiological data indicating temporary inhibition of olfac-tory perception after MFM.

Introduction

Olfactory sensory epithelia are unique among the sensory epithelia with primaryneurons in that their neuronal cells are continuously replaced (e.g., Moulton,1975; Graziadei and Monti Graziadei, 1978a). A number of techniques (e.g.,olfactory bulb ablation, olfactory nerve sectioning, destruction of the sensoryolfactory epithelium by ZnSGyirrigation, and blockade of mitosis by colchicine)have been applied to study the turnover rate of the receptor cells and indicate thatthe replacement of olfactory sensory cells of adult warm blooded vertebrates oc-curs about every 30 days (Clark and Warwick, 1946; Sen Gupta, 1967; Albertsand Galef, 1971; Takagi, 1971; Powers and Winans, 1973; Matulionis, 1975;Breipohl etal., 1976; Harding and Wright, 1979; Monti Graziadei and Graziadei,1979; Booth et al., 1981; Simmons and Getchell, 1981a, 1981b; Simmons et al.,1981). In fish and tiger salamanders the time span between olfactory bulb abla-tion or olfactory nerve sectioning and complete reconstitution in the populationof receptor cells in the olfactory epithelium seems to be considerably longer thanin mammals (Breipohl and Ohyama, 1981; Simmons and Getchell, 1981a, 1981b;Simmons et al., 1981). Nevertheless, 7 days following the ablation of the olfac-tory bulbs of goldfish, at least a temporary enhancement of cell mitosis has beennoted (Breipohl and Ohyama, 1981). The question of whether the rapid cellrenewal within the olfactory epithelium is due to the stimulation of quiescentand/or normally and continuously dividing progenitor cells is not clear. To helpanswer this question, studies on the epithelial composition of a layer of suppor-ting cells, sensory cells, and basally located progenitor cells were undertaken inthe present work under normal and experimental conditions.

Preliminary investigations on the effect of N-methyl-formimino-methylester

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(MFM) on electrophysiologically measured olfactory thresholds of mice sug-gested to us that MFM may cause degeneration of olfactory sensory cells and thatcell degeneration may be responsible for the obtained temporary inhibition ofolfactory perception (Schmidt et al., in preparation). In the present study, themorphological alterations in the olfactory sensory epithelium of mice after MFM-application are described. Special attention has been paid to the time course ofolfactory nerve cell alteration and to changes in the overall composition of thesensory epithelium.

Methods

White adult male mice (NMRI strain) served as experimental animals. As describ-ed in detail elsewhere (Schmidt et al., 1981), saturated gaseous MFM was applied,with the help of a syringe, at room temperature, for approximately 0.5 s (2 — 3 in-halations). In addition to the controls, 3 experimental animals were sacrificed ateach of 8 intervals after the application of MFM (1.5 h; 1, 3, 7, 14, 21, 35 and 56days). The nasal cavity was superfused with a 0.1 M 3% glutaraldehyde solutionin sodium cacodylate buffer (pH 7.4) at room temperature for 3—5 min. Afteropening the nasal cavity, the olfactory epithelium was fixed for another 24 h at4°C in the same fixative. The sensory epithelia were dissected out, washed in 2%sucrose in sodium cacodylate buffer (pH 7.4), postfixed in buffered 1%osmiumtetroxide (pH 7.35), dehydrated, and finally embedded in Epon. The lightmicroscopical investigations were performed on 1 — 2 /im sections stained with1 % azure II-methylene blue. In the experimental animals, 3 subsequent 500 /xmpieces of olfactory epithelium were sectioned and every 20th section of such aspecimen block was used for semiquantitative counting of mitoses (Diagram 2).The counts of mitoses were calculated per 100 /tin2.

Results

As in other mammalian species (Moulton and Beidler, 1967; Moulton, 1975b),the olfactory epithelium of mice is mainly composed of sensory cells, supportingcells, and basally located progenitor cells. The most apical compartment of theepithelium contains a peripheral zone relatively free of nuclei and a single layer ofsupporting cell perikarya, together accounting for about 1/7 to 1/3 of the heightof the epithelium (Figure 1; Diagram 1). Beneath this apical compartment is thebroad layer of sensory cell perikarya comprising about 2/3 of the epitheliumheight. Sensory cell perikarya often show a columnar arrangement. Necroticnucleic occur only rarely. From the sensory cell perikarya, dendrites extend up tothe epithelium surface and end in the olfactory knobs. A distinct cellular com-partment exists basal to the sensory cell perikarya, and comprises 1/10 to 1/6 ofthe height of the epithelium. The most prominent feature of this small band arethe flat, elongated dark-staining basal cells which form a thin, more or less con-tinuous layer immediately above the basement membrane. Interspersed betweenthese typical basal cells and the most basally located sensory cell perikarya arelighter staining cells with a taller perikaryon, the so-called blastema cells (Andres,1965) or globose basal cells (Graziadei and Monti Graziadei, 1978b), which hereinare called progenitor cells since they are precursors for olfactory receptor cells

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Fig. 1. Olfactory epithelium of control mouse. SCL: apical epithelial compartment with supportingcell perikarya; NL: layer of sensory cell perikarya; PCL: compartment of progenitor cells.

(Andres, 1965; Graziadei and Monti Graziadei, 1978b). Mitoses are regularlyobserved in the layer of progenitor cells, and rarely in the layer of supporting cellperikarya. Mitotic figures have been observed only exceptionally in the middle ofthe epithelium occupied by the perikarya of the sensory cells and the interspersedduct cells of Bowman glands.

When compared with the controls, no gross morphological alterations can beseen in the sensory cell layer of the epithelium 1.5 h and 24 h after the applicationof MFM (Figures 2 and 3). However, mitotic figures in the progenitor cell layeroccur more frequently (Diagram 2). The demarcation between the progenitorcells and the most basally located sensory cell perikarya is less sharp than in con-trols. Three days after MFM application no obvious morphological alterationsare observed in the supporting cell layer, but enlarged numbers of pycnotic cellsappear in the sensory cell layer (Figure 4). The compartment of the progenitorcells is enlarged. Occasionally distinct irregular indentations of the blastema cellband into the layer of the sensory cell perikarya are observed. The number ofmitotic figures in the layer of progenitor cells exceeds that in the controls, but islower than that 24 h after MFM application (Diagram 2). Moreover, a discon-tinuation in the thin band of the typical basal cells is repeatedly observed. Ir-regular shapes of these cells, rather than flat and elongated configurations as inthe controls, become more frequent.

Seven days after MFM application the morphological alterations in the sensoryepithelium are obvious (Figure 5). Though the overall composition of theepithelium is not altered, one finds more conspicuous indentations of the pro-genitor cell layer into the sensory cell layer and a more irregular demarcation bet-ween the sensory cell layer and the compartment of the progenitor cells (Diagram1). After 7 days, the number of mitotic figures is greater than in all earlier stages(Diagram 2). Flat basal cells only occur infrequently (Figure 5). The mor-phological appearance of the layer of supporting cell perikarya is unaffected.

The morphological alterations in the overall composition of the sensory

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Diagram 1. Schematic representation of MFM-induced changes in the overall epithelium composi-tion of an apical compartment with supporting cell perikarya (hatched), a middle layer of neuronalperikarya (dark), and a progenitor cell compartment (white) at different survival times (1.5 h —56days). Subepithelial connective tissue is stippled. Two examples are drawn per survival time.

epithelium are more pronounced 14 and 21 days after the application of MFMand the height of the epithelium is considerably reduced. Only a thin band ofreceptor cell perikarya is typically observed between the supporting cell layer andthe increased compartment of progenitor cells (Diagram 1). The dark-stainingbasal cells, if present at all, are often triangular in shape and have apical exten-sions (Figures 6, 7 and 8). The number of mitoses is higher than at earlier stagesand reaches values which are about 5 to 6 times those of the controls (Diagram 2).Mitotic Figures are not restricted to the direct vicinity of the basement membranebut appear also deeper in the layer of blastema cells.

Five weeks after exposure to MFM, the morphological alterations are less con-spicuous than before and the samples again show structural features resemblingthose of the controls (Diagram 1). However, signs of thinning out of the sensorycell perikarya compartment and an irregular demarcation between this cellular

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Fig. 2. Olfactory epithelium of mouse 1.5 h after exposure to MFM. Demarcation between thelayers of sensory and progenitor cells is less clear than in Fig. 1.Fig. 3. Olfactory epithelium of mouse one day after exposure to MFM. Demarcation between pro-genitor cell compartment and neuronal cell compartment is not sharp. M: mitosis.

[number of mitotic figures per (100 um)2]

21 28Survival Time

Diagram 2. Rate of mitosis in the progenitor cell compartment of mouse olfactory epithelium at dif-ferent survival times after exposure to MFM. C: control. The bars indicate the standard deviations.

band and the compartment of progenitor cells can still be observed. In the latter,the thin flat layer of typical dark staining basal cells reappeared almost complete-ly. The rate of mitoses decreased considerably and is lower than in all thepreceding experimental stages. Nevertheless, it is still higher than in the controls(Diagram 2). However, samples with a clearly reduced number of olfactory knobsat the epithelial surface can still be detected.

Eight weeks after MFM exposure no obvious structural differences can be seenin the olfactory sensory epithelium when compared with controls (Diagram 1;

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Fig. 4. Mouse olfactory epithelium 3 days after exposure to MFM. No principal differences in com-parison to controls. BL: light staining "globose basal cells". Note row of typical dark staining basalcells (arrows).

Fig. 5. Mouse olfactory epithelium 7 days after exposure to MFM. No sharp demarcation exists bet-ween neuronal cell layer and progenitor cell compartment. M: mitosis.

Figure 9). The number of mitoses decreased further but is still slightly higher thanin untreated animals (Diagram 2).

Discussion

1) MFM, a highly volatile substance, causes degenerative alterations in theolfactory epithelium of the nasal septum of mice.2) Only sensory cells show degeneration after the above described treatmentwith MFM.3) In addition to the necroses of receptor cells and the irregular thinning out ofthe receptor cell perikaryal layer, morphological alterations are observed in theprogenitor compartment. Degenerative and reactive alterations become less evi-dent from 5 weeks onwards after MFM exposure.4) The rate of mitoses increases a few hours after MFM treatment, thendecreases (3 days) and then increases again to reach values 5 or 6 times that ofcontrols after about 3 weeks. After 5 weeks, the rate of mitoses gradually ap-proaches that of the controls.5) A temporal blocking of olfactory function after MFM application, as sug-gested from electrophysiological investigations (Schmidt eta I., 1981), may be dueto the morphological alterations described here. However, nothing can be saidabout the mechanism of degeneration due to MFM.

The function of the olfactory sensory epithelium is related to an exposure toairborne chemicals. These chemicals may have to pass through the mucus embed-ding the receptive structures. However, airborne chemicals have not been used as

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Figs. 6 and 7. Mouse olfactory epithelium 14 days after exposure to MFM. The layer of neuronalcell perikarya is markedly reduced. The progenitor cell compartment is enlarged due to an accumula-tion of globose basal cells. The demarcation between these compartments has an irregular course.Dark staining basal cells occasionally show transition forms to somewhat triangular shaped cells(thick arrows). Thin arrows indicate mitoses. Note the reduction of olfactory knobs at the epitheliumsurface.

yet to study the well established regenerative capacity of the receptor cells at amorphological level. Many airborne chemicals are known to cause inhibition ofolfactory perception. Among these substances MFM is a very potent olfactoryblocker (e.g., Herberhold, 1975). Previous investigations showed that an ex-posure of only 0.5 s to MFM causes a considerable blocking of the elec-trophysiological activity in the olfactory bulbs of mice as inferred from the EEG.The olfactory threshold of geraniol, for example, increased by a factor of 106

after MFM application, was maintained at this level of 3 weeks and only becamenormal from 5 weeks onwards (Schmidt et al., 1981). Moreover, MFM causescomplete anosmia upon inhalation (Effenberger, personal observation). Asdemonstrated herein, MFM may act as a rather specific toxic substance to theolfactory receptor cells. In spite of massive degeneration of the sensory cells bet-

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Fig. 8. Mouse olfactory epithelium 21 days after exposure to MFM. Similar epithelium configura-tion as 14 days after exposure (c.f. Figs. 6 and 7). Note triangular-shaped dark-staining basal cells(thick arrows). M: mitosis.

ween one and 3 weeks after MFM exposure, the other cellular components of theolfactory epithelium remained light microscopically unaffected (supporting cells)or showed an increased activity (progenitor cells). The above mentioned elec-trophysiological data can therefore be explained by a reduced number of receptorcells being active from one week onwards. These data match the elec-trophysiological data (Schmidt etal., 1981). A comparable relationship between afunctional recovery and a morphological repair of the olfactory epithelium hasbeen reported for the tiger salamander (Simmons and Getchell, 1981a, 1981b;Simmons et al., 1981) after olfactory nerve section.

However, cessation of the electrical activity of the olfactory bulb and an in-crease of the thresholds for different odorants (unpublished) occurs several hoursafter MFM treatment (Schmidt et al., 1981). This indicates that, in spite of arather unchanged morphology at the level of the light microscope, the sensorycells are already affected in this early phase. It is very likely that a toxic effect ofMFM on the membranes of the sensory cell terminals leads only gradually tonecrosis of receptor cells. Further transmission electron microscopic investiga-tions may support this hypothesis.

As well as reduction of the sensory cell compartment, a considerable increaseof the progenitor cell compartment was found between 3 days and 5 weeks after

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Fig. 9. Mouse olfactory epithelium 56 days after exposure to MFM. No principal differences can beseen between these and the controls.

exposure to MFM. This was mainly due to an increase in the number of "globosebasal cells" (Graziadei and Monti Graziadei, 1978b). At the same time, the layerof dark-staining basal cells became discontinuous and their shapes showed transi-tional forms to blastema cells. From this it was concluded that these dark, in-termediate, and light basal cells may have four different pathways but may stillhave a common ancestor (Diagram 3) i.e., (i) they are on their way to differen-tiate into receptor cells; (ii) they are typical blastema cells and continue with celldivision; (iii) they are daughter cells, which differentiate into the flat andelongated dark-staining basal cells; or (iv) they represent transitional stages bet-ween such dark-staining flat and elongated basal cells and dividing cells.

Moulton and Fink (1972) estimated that about 90% of the basal daughter cellsmigrate to the periphery and differentiate into receptor cell neurons and suppor-ting cells, while about 10% continue with mitotic activity. The present resultssupport these findings and those of Andres (1965) related to the progenitor func-tion of blastema cells, and suggest that some of the progenitor cells do not takepart in the normal course of the receptor cell generation cycle but behave as quies-cent cells.

The decline in the rate of mitoses around day 2 after MFM-exposure (Diagram2) may be explained by a temporary exhaustion of mitotic-active blastema cellsdue to a pronounced transformation of their daughter cells into receptor cells(c.f. Breipohl and Ohyama, 1981). From 3 days onwards the progenitor cells,which up to the MFM-treatment have been in a "quiescent stage" (c.f. Leblondand Walker, 1956; Moulton, 1975b), reenter the regeneration cycle and par-ticipate in the increase of mitotic activity (Diagram 2). The lighter staining nucleiof the transitional cells indicate despiralization of the chromatin and a higher

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Dark stainingbasal cells

Light" staining (globose)

basal cells

^V/

Sensory cells

Comparhnenr ot

progenitor cells

Comparrmenr of

matured cells

Diagram 3. Hypothetic transformation pathways between progenitor cells and olfactory receptorcells. The origin of new supporting cells is not included because it is not mentioned in this paper.

metabolic activity, as is necessary, for example, for DNA-reduplication in theS-phase of the generation cycle and later on for mitosis. Once a nearly normalpopulation of sensory cells has reestablished, quiescent dark-staining basal cellsshould reappear, as seen in our material from 5 weeks onwards after MFM ex-posure.

The regulatory mechanism for establishing a constant number of sensory cellsin the olfactory epithelium is presently not understood. It is possible that the sizeof the receptor cell population is only limited because of the fact that individualreceptor cells die after a species-specific life span. Some authors assume thatunder normal conditions there are matching rates of basal cell division and ofsensory cell necrosis (Moulton, 1975b; Simmons and Getchell, 1981b). Extendeddying of the receptor cells may lead to an enhanced mitosis and overhastytransformation of progenitor cells into sensory cells (Monti Graziadei, 1979;Breipohl and Ohyama, 1981). Different levels of steroid hormones may also af-fect mitosis and transformation of progenitor cells as reported by Balboni andVannelli, 1981, who found not only considerable differences in basal cellnumbers between male and female rodents but also changes during the course ofthe ovarial cycle. Moreover, Vannelli and Balboni (1982) and Stumpf and Sar(1982) have evidence that the olfactory epithelium of rodents serves as a targetorgan for estrogens. Whether cyclic changes in olfactory sensitivity (Schmidt,1978, 1979) in mice may be correlated with cyclicity in cell turnover of sensorycells needs further study.

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Acknowledgements

With support of: A B M - Massnahme Nr.: la - 2-5100 / D la 312 - 5100/ lfd.Nr.: 243/79 and, in part, the Deutsche Forschungsgemeinschaft.*Reprint requests to Prof.Dr.W.Breipohl, Institut fur Anatomie, Universitat-sklinikum Essen, Hufelandstr. 55, 4300 Essen 1, FRG.

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